Secretion, purification and activity of two recombinant pepper endo-beta-1,4-glucanases expressed in the yeast Pichia pastoris.
Two pepper endo-beta-1,4-glucanases, involved in fruit softening (cCel1) and leaf and flower abscission (cCel2), have been expressed in Pichia pastoris. Secretion was obtained by using either the mouse alpha-factor signal (cCel1) or the native signal sequence (cCel2). Times for optimal expression of the two proteins were different and cCel2 appeared very sensitive to proteolytic degradation. A one-step purification protocol yielded cCel2 in a pure form, while an additional chromatography step was necessary to purify cCel1. The two recombinant proteins are highly active and able to degrade carboxymethylcellulose in viscometric assays. Moreover, they have both a molecular mass (54 kDa) and an isoelectric point (7.2 for cCel2 and 8.5 for cCel1) equal to those of the native proteins, thus suggesting that post-translational modifications have properly occurred.
Ferrarese
L
L
Department of Biology, University of Padua, Italy.
Trainotti
L
L
Gattolin
S
S
Casadoro
G
G
D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002480:Cellulase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression / Q000235:genetics; D007526:Isoelectric Point; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D007527:Isoenzymes; D010940:Plant Proteins; D011994:Recombinant Proteins; D002480:Cellulase; C090774:endoglucanase EGL1; D002266:Carboxymethylcellulose Sodium